Project Summary We seek to identify the one or more Cytochrome P450s (CYPs) responsible for metabolism-mediated resistance to pyrethroid insecticides in Aedes aegypti, an important vector of human diseases such as dengue and Zika. Pyrethroid insecticides are widely used for control of adult Aedes, consequently resistance has evolved in many populations, representing a global challenge for control of this vector and the diseases it transmits. Finding strategies for delaying the development of resistance by mosquito vectors to the few available insecticides is critical. To do so, it is essential to identify the cause of resistance in order to design sensitive and precise monitoring programs, to understand the population genetics and the evolution of resistance, and to design effective countermeasures to slow the development of resistance. For CYP- mediated resistance, measurements of protein levels and non-synonymous polymorphisms are ultimately the most important parameters to measure. Our long term goals are to develop the necessary technologies to monitor and study resistance alleles so that this important evolutionary process can be understood and so that the development of resistance can be delayed. Our short-term goal is to identify the mutations responsible for pyrethroid resistance in the highly resistant Singapore (SP) strain of Aedes aegypti. To accomplish this goal we will pursue three specific aims. Aim 1. Quantify the levels of CYP proteins present in the microsomes from susceptible and resistant strains and determine whether the overexpression is genetically linked to resistance. Aim 2. Determine if there are any non-synonymous polymorphisms in the ~160 CYPs and whether they are genetically linked to resistance. Aim 3. Validate the role of the one or more CYPs identified in Aims #1 and #2 in pyrethroid resistance. These results will provide us with an unprecedented understanding of pyrethroid resistance in A. aegypti, identify genes worthy of future study and provide the fundamental basis for resistance monitoring and management in this important vector of human disease.